BERR RB ERE REE EB ESSERE EE CHAPTER 9 Salmonella FDA DETECTION PROTOCOL; ELISA TECHNIQUE; DNA PROBE TECHNIQUE nei INTRODUCTION Properties ‘The genus Salmonella belongs to the family Enterobacteriaceae. As the name implies, Enterobacteriaceae is comprised of bacteria that proliferate in the intestine. Several genera of this family include pathogenic species In addition to Salmonella, Enterobacteriaceae includes food-trensmitted pathogenic species of these genera: Escherichia, Shigella, and Yersinia. ‘Like other Enterobacteriaceae genera, Salmonella is a gram-negative flagellated rod-shaped bacterium, Salmonellae are facultative anaerobes with both respiratory and fermentative metabolic pathways. They are oxidase negative, ferment glucose dad produce acid and gas, grow on citrates as a sole source of energy, decarbory- late lysine and ornithine, generally produce hydrogen sulfide, and do not hydrolyze urea. One of the characteristics of this genus is that most members do not ferment lactose of sucrose. Classification and Nomenclature Based on their reaction with specific antibodies, Salmonella isolates can be classi- fied into ~2400 Salmonella serovars. This classification is based on the type of antigens produced by isolates. Somatic (O) Antigens ‘The O antigens are associated with the lipopolysaccha- rides (LPS) on the external surface of the bacterial outer membrane. Somatic ‘entiyons are heat stable and resistant to alcohol and dilute acids. Fock Mcrbiniogy, By blared E.Yousel an8 Carclys Cartstrom . SEN OAN2AVED Copyright © 20D by Jobs Wiley & Sons, Ine. 167 Escaneado con CamScanner168 Salmonella Flagellar (H) Antigens These are the antigens associated with the peritrichous flagella. Flagellar antigens are heat-labile proteins. Capsular (K) Antigens These antigens are produced by Salmonella serovars that produce capsular material. Capsular antigens are heat-sensitive carbohydrates. Salmonella serovars cannot be differentiated biochemically. The Kauffmann— White scheme for classifying salmonellae assigns a species status to each serovar. ‘Therefore, differentiation between Salmonella typhimurium and S. enteritidis, for example, is based on serotyping. According to a more recent classification scheme, serovars are not different species because they have 270% DNA homology, and numerical taxonomy supports their similarity. Therefore, two Salmonella species only are now recognized by the CDC: Salmonella enterica and Salmonella bongori. According to this scheme, the vast majority of the serovars are under S. enterica, with only 20 serovars belonging to S. bongori. Under this scheme, what used to be S. typhimurium is now S. enterica serovar Typhimurium or simply Salmonella ‘Typhimurium, This latter scheme will be followed in this chapter. The Disease Salmonella is an invasive bacterium that causes human infection, known as salmo- nellosis. This disease is widely spread in many countries and affects the young and the elderly the most, Although it is assumed that Salmonella infectious dose is high, infection may result from ingesting as little as 1-10 cells. There are different syndromes of human salmonellosis. Typhoid (enteric) fever is caused by Salmonella Typhi and Salmonella Paratyphi. It accounts for <5% of cases of salmonellosis. Symptoms include diarrhea, abdominal pain, headache, and ~— prolonged high fever. The incubation period varies from 1 to 7 weeks, and disease may last 1-8 weeks. The most common form of human salmonellosis is the Salmo- nella gastroenteritis (or enterocolitis). This is caused by at least 150 serovars of Salmonella. Symptoms of Salmonella gastroenteritis include diarrhea, abdominal pain, chills, moderate fever, vomiting, dehydration, and headache. Incubation period is 12-36hr, and duration is 1-4 days. Foods Implicated Since the intestine is the natural habitat of Salmonella serovars, raw foods of an animal source occasionally harbor the pathogen. Salmonella, therefore, is found in poultry products, including chicken, eggs, and turkey. Shellfish, milk, salads, and can- taloupes also have been implicated in salmonellosis outbreaks. Water has been a vehicle for transmission of salmonellosis. Workers who do not observe proper per- sonal hygiene, especially those working in food harvesting, processing, and service, are potential sources of food contamination with Salmonella. The most common serovar-associated food-transmitted salmonellosis are caused by Salmonella ‘Typhimurium and Salmonella Enteritidis. Most Salmonella serovars grow only in the mesophilic temperature range; however, a few serovars can grow at temperatures as low as 2-4°C or as high as 54°C, Foods with pH < 4.5 do not normally support the growth of Salmonella, but Escaneado con CamScannerINTRODUCTION — 169 Some serovars grow at pH 4.0 (c.g,, Salmonella Infantis). Pasteurization tempera- tures readily inactivates Salmonella serovars. One serovar (j.e., Salmonella Senftenberg 775W), at least, is known to have exceptional resistance to heat. Recent appearance of isolates with multidrug resistance (e.g., Salmonella Typhimurium DT 104) is a potential threat to the safety of consumers and raises a great health concern worldwide. Detection Some Salmonella serovars are known to be pathogenic to humans, but all salmo- nellae are unacceptable in ready-to-cat foods. It is uncominon to find Salmonella in countable levels in food. Therefore, food is analyzed for the presence or absence of Salmonella, and detection methods rather than direct plating are used. Conventional methods for detection of Salmonella in food are based on the cultural, biochemical, and serological properties of the microorganism. Alternative rapid methods are becoming popular in the food industry, and most of these are based on genetic or immunoassay techniques. These methods do not include the labor-intensive and time-consuming biochemical identification steps. ‘The rapid methods still require sample enrichments, and only the negative results are considered conclusive, Positive results by rapid methods require verification using the conventional biochemical and serological identification techniques. In lab- oratories where a large number of samples are analyzed routinely, these alternative methods are useful in rapidly screening these samples for Salmonella. Since most ‘commercial food samples are expected to be Salmonella free, use of rapid methods as a screening tool could save time and efforts. Conventional Methods Conventional methods of detection of Salmonella in food depend on the cultural, biochemical, and serological properties of this microor- ganism. Typical Salmonella isolates are those that (i) produce acid from glucose, but not from lactose or sucrose, in triple sugar iron (TSI) agar medium; (ii) decarboxy- late lysine to cadaverine (alkaline product) in lysine iron agar (LIA) medium; (ii) generate hydrogen sulfide (H,S) in TSI agar and LIA; (iv) do not ferment lactose or sucrose in xylose lysine desoxycholate (XLD), Hektoen enteric (HE), and similar agar media; and (v) do not hydrolyze urea, Although Salmonella’s inability to ferment lactose or sucrose is an important defining biochemical property, some ‘Salmonella isolates are lactose or sucrose positive, Fermentation of these sugars is coded on a plasmid that may be acquired or lost through conjugative genetic ‘exchange. Conventional methods involve (a) preenrichment and selective enrichment, (b) isolation steps, and (c) identification tests. Enrichment and isolation steps are primarily cultural techniques, whereas identification relies on biochemical and serological testing. Enrichment Unlike clinical samples, foods contain only a small population of athogens, if any. While isolation of Salmonella from clinical samples often requires Ply direct plating on selective agar media, detection of the pathogen in food neces- Sitates an enrichment process to increase the numbers before isolation and identi- fication can be accomplished. Salmonella in food is often present in an injured state. Cells are often injured during food processing (e.g,, acidification and dehydration) Escaneado con CamScanner170 saimonetia and storage (c.g,, freezing). Enrichment steps are normally designed to resuscitate these injured cells, Preenrichment and selective enrichment steps are carried out to resuscitate injured salmonellae and increase their number to detectable levels. In the preen- tichment step, the food sample is mixed with a suitable nonselective medium and the mixture is incubated. This results in a modest increase in Salmonella population and appreciable increases in numbers of competing microbiota in the sample. After the preenrichment, it is presumed that salmonellae are healthy enough to endure a subsequent enrichment step using media containing selective agents. Therefore, a small volume of the preenriched sample is transferred into the sclective enrichment medium, This selective enrichment step allows growth of Salmonella and suppresses competing microbiota. Media used in preenrichments vary with the type of food analyzed. Lactose broth is the most commonly used preenrichment medium, in spite of the fact that most Salmonella isolates do not use lactose as a carbon source. This medium, however, seems suitable for the slow recovery of injured cells in the food sample. Rich food such as dry milk may be enriched for Salmonella by simply mixing the sample with distilled water and incubating the mixture. In contrast, nutritionally poor foods (e.g, spices) are preenriched in Salmonella by adding a rich microbiological medium such as trypticase soy broth. Selective enrichment includes subculturing the preenrichment into tubes con- taining selective broth. Selenite cystine broth and tetrathionate broth are widely used for selective enrichment. Composition and selective properties of these two media are reviewed later in this chapter. Rappaport-Vassiladis medium has been recommended as a replacement for the selenite cystine broth, It is advisable to use two or more selective enrichment media to improve the recovery of Salmonella from the sample. Isolation Isolation includes streaking enrichments onto selective-differential agar media and recognizing presumptive Salmonella colonies on the incubated plates. ‘These isolated colonies are subcultured for subsequent identification. Media for Salmonella isolation should contain selective agents such as bile or desoxycholate salts, brilliant green, and bismuth sulfite. These agents inhibit gram-positive and nonenteric bacteria, Differentiation of Salmonella and non-Salmonella bacteria is accomplished by inclusion of suitable carbohydrate/pH indicator combinations. Lactose, sucrose, and salicin are not typically fermented by Salmonella, and thus production of acid from these carbohydrates indicates non-Salmonella isolates. If lysine is present in the medium, Salmonella decarboxylates the amino acid, pro- ducing alkaline products that change the color of the pH indicator in the agar sur- rounding the colony. A familiar differential system in these media depends on the ability of Salmonella to release H,S from sulfur-containing substrates (e.g,, sodium thiosulfate) using its desulfhydrase. In addition to the enzyme substrate, the isola- tion media are formulated to contain water-soluble ferrous or ferric salt which reacts with the released H,S to produce a black precipitate in and around the typical Salmonella colonies. ‘The principles just described were implemented in formulating several isolation media. Xylose lysine deoxycholate (XLD) agar and Hektoen enteric (HE) agar are commonly used for isolation of Salmonella from enrichments. Additionally, bismuth sulfite (BS) agar is an ideal medium for isolating salmonellae. It has high selec- —i Escaneado con CamScannerORGANIZATION 171 tivity for the bacterium and allows detection of small levels of H2S generated by isolated colonies. It is common to streak a Salmonella-enriched sample onto two or more of these selective-differential agar media; thus, recovery of pathogens from the food is improved and method sensitivity increases. Description and properties of these media are discussed later in this chapter. Biochemical Identification Conventional detection methods rely on biochemical tests to identify Salmonella, Decarboxylation of lysine in LIA, fermentation of glucose anaerobically in TSI agar, production of H,S in TSI agar and LIA media, and utilization of citrates are important biochemical properties for identification of Salmonella isolates, Inability of Salmonella to (a) hydrolyze urea, (b) produce indole from tryptophan, and (c) grow in potassium cyanide broth are also useful bio- chemical tests for the characterization of the microorganism. Biochemical identifi- cation of Salmonella, therefore, requires testing presumptive isolates in TSI agar and LIA and running additional biochemical tests. Serological Identification/Confirmation Isolates that produce typical reactions by biochemical testing should be confirmed as Salmonella by serological analysis. If iso- lates react with antibodies developed against Salmonella’s somatic or flagellar anti- gens, this confirms the isolates as Salmonella. Isolates, therefore, are examined by a polyvalent flagellar test and a polyvalent somatic test; Salmonella produces agglu- tination upon testing, For serotyping, a more elaborate scheme of serological analy- sis is needed. Rapid Methods Rapid alternative detection methods for Salmonella require enrichment steps similar to those used in the conventional protocols. Additionally, a postenrichment step may be required to condition the isolates for the rapid tech- nique. Time savings are achieved when these rapid methods are applied to screen a large number of samples and exclude the Salmonella-negative ones (Fig. 9-1). Positive results by the rapid methods still require confirmation by conventional thods for detection of Salmonella in food are based on techniques. Rapid met immunological or genetic testing. Immunoassay-based and DNA hybridization- based methods are included in this chapter. OBJECTIVES + Detect Salmonella in food using a conventional method (the FDA protocol). + Apply alternative rapid detection methods using molecular and serological techniques. + Understand the merits and limitations of different detection methods. « practice safe handling of pathogens in the laboratory. ORGANIZATION aboratory exercises to detect Salmonella in food are presented in this ig the first exercise, students analyze food using a conventional as rapid detection methods are used in the second and third exer- ‘Three laborat chapter. Duriny method, wheres Escaneado con CamScanner172 salmonetia ° Proenuichmen {Lactose broth) 26 { ‘Soleotive enrichment {SC broth, TT broth} Time elapsed (hy) Isolation estenichment [88, HE, XLD agars) IM broth (ELISA) or GN broth (ONA probe)] 6 i test g tal TELISA oF DNA probe} | | coreiaty 7 Biochemical ‘soreening ITSIA, LIA} poe Te whet complete i Gontrmaion _ i T i 1 1 v Serotyping Fig. 9.1. Detection of Salmonella by conventional and rapid methods. Time schedule shown may be different than that followed in the laboratory exercisesit represents a minimum time frame. Labels of y axis: [A] time to complete a negative rapid test (~63hr), [B) time to com. plete a negative conventional test (~96hr). cises (Table 9.1). The conventional method is completed in five laboratory periods while each of the rapid methods requires four periods. These three exercises may be run sequentially or simultaneously. Since the three methods share common enrichment steps, it saves time to run all three exercises side by side. In this case, however, provisions should be miade not to run the ELISA and DNA probe steps in the same laboratory period because of the complexity of these techniques com- pared to the cultural protocol. Procedural details of each exercise are presented later in the chapter. Escaneado con CamScannerREFERENCES 173 TABLE 9.1, Proposed Schedule’ for Dete of Salmonella in Food Using Conventional (Culture-Blochemical-Serolo, 1) and Rapid Methods . Immunoassay-Based Period Conventional Method? Method" Genetic-Based Method’ 1 Preenrichment Preenrichment (in LB) (in LB) 2 Selective enrichment Sclective enrichment _ Selective enrichment (in TT and SC) (in TT and SC) (in TT and SC) 3 Isolation Postenrichment Postenrichment (on BS, HE, and XLD agar) (in M broth) (in GN broth) 4 Biochemical identification ELISA test DNA probe test (on TSI agar and LIA slants) 5 Inspection of TSI agar and LIA slants* * If the class does not meet daily cultures that require only 24h of incubation will be refrigerated until 24h before the class meets and then incubated. ® Tfall three methods are executed simultaneously, ELISA and DNA probe tests may not be run during. the same laboratory period because of the complexity ofthese techniques. « Slants should be inspected after no longer than 24h of incubation. Holding slants for additional incu- ation or in the refrigerator allows the spread of the black precipitation reaction, which makes it diffi- ‘cult to determine the result of the test. PERSONAL SAFETY All Salmonella serovars are considered pathogenic to humans and thus should be handled with care, Follow the safety guidelines that are reviewed at the beginning of this manual. Use disposable gloves when handling the isolates and the pathogenic cultures. Make sure the work area is sanitized after use. REFERENCES Andrews, W. H., and T. S. Hammack. 2000. Bacteriological Analytical Mannual Online, Chapter 5: Salmonella. Available: http: //www.cfsan. fda .gov/~ebam/bam_ toc.html. “Andrews, W.H., R. S, Flowers, J.illiker, and J. S, Baily, 2001, Salmonella. In F., Downes and K. Ito (Eds.), Compendium of Methods for the Microbiological Examination of Foods, 4th ed. (pp-357-380). American Public Health Association, Washington, DC. Centers for Disease Control and Prevention (CDC). July 2002. Salmonella enteritidis. ‘Availablesht tp: //www.cde.gov/neided/dbnd/diseaseinfo/salment_g.htm. prAoust, J-Y, J. Maurer, and J. S. Baily. 2001. Salmonella Species. In M. P. Doyle, L. R. ‘Beuchat, and T. J. Montville (Eds.), Food Microbiology, Fundamentals and Frontiers, 2nd ba. (pp. 141-178). American Society for Microbiotogy, Washington, DC. Difeo, 1998. Difco Manual, Lith ed. Difco Laboratories, Sparks, MD. Escaneado con CamScanner174 Sammonota EXERCISE 1: CONVENTIONAL METHOD PROCEDURE OVERVIEW In this laboratory exercise, presence of Salmonella in food will be tested using a | modification of the method described in the FDA Bacteriological Analytical Manual (BAM, 2000). The method depends on cultural, biochemical, and serological tech niques and will be referred to as a “conventional/BAM" or simply “conventional” method. Two alternative rapid methods are also described later in this chapter as separate exercises, Since similar enrichment steps are applied in the three methods, it is advantageous to run two or all three methods simultaneously. ‘The conventional/BAM method includes five major steps: (i) preenrichment, (ii) selective enrichment, (iii) isolation on selective-differential agar media, (iv) identi- fication and confirmation by biochemical testing using differential media, and (v) serological tests In this laboratory exercise, the biochemical confirmation and sero- logice! testing will not be completed. An overview of this method, as applied in this laboratory exercise, is shown in Figs. 9.1 and 9.2. The detection by this conventional ' method will be completed in five laboratory periods, which correspond to four of the five steps just indicated (Table 9.1). Results Interpretation Positive results by the conventional detection method, as applied in this exercise, require that 1. Salmonella multiplies in the preenrichment (lactose broth) to a high enough level that some viable cells are transferred into the selective enrichment. 2, Salmonelia is present in at least one of the selective enrichment tubes (SC or TT broth tubes) at a level suitable for transfer onto isolation agar media by streaking. 3. Enrichments result in a relatively high proportion of Salmonella to contami- } nants so that streaking on isolation agar plates (BS, HE, or XLD) produces at least one isolated colony with typical Salmonella characteristics on any of these plates. 4, One isolate at least, from the isolation agar plates, produces typical biochem- ical reactions of Salmonella on TSI agar and LIA. MEDIA Reactions of typical Salmonella isolates on these media are summarized in Table 9.2. Media composition is provided in Appendix C. Bismuth Sulfite (BS) Agar ‘This is a selective-differential medium used for the isolation of potential Salmonella colonies. Brilliant green in the medium acts as a selective agent by inhibiting the growth of gram-positive organisms. Bismuth sulfite has both selective and differen- ee Escaneado con CamScannerCONVENTIONAL METHOD 175 41. Preenrichment (in bottes oF stomacher bags) 1 cubation at 95°C, 24 hr 2, Selective enrichment sc 7 Tncubation at 35°C, 24 hr 8. Isolation GD 4 @ xD BS 1 Incubation at 35°C, 24 hr 4, Biochemical screening TsI uA a Tncubation at 35°C, 24 hr ¥ '5, Biochemical confirmation/serotyplng Fig. 9.2. Conventional method for detection of Salmonelta in food. tial effects, The bismuth selects against coliforms and also against gram positives. ‘Tie sulfite allows differentiation of organism producing the enzyme desulfhydrase. Froduction of desulihydrase by Salmonella results in formation of hydrogen sulfide (HS) from the sulfite. The ‘H,S formed then reacts with ferrous sulfate (FeSO,) to (i) lack precipitate of ferrous sulfide (FeS), Salmonella colonies on BS agar ‘Orpen black to green in color with or without a dark helo in the surrounding agar aed the colonies often appear to have a metallic sheen. Hektoen Enterle (HE) Agar Hiektoen enteric agar is a selective-differential medium used for the isolation of Hesumptive Salmonella colonies. Bil salts bromothymol tes nodiuead aussi aes te that inhibit gram-positive bacteria. Acid fuchsin and bromothymol selective agent® cree eae blue also function as acid indicators detecting acid production from the fermenta- Escaneado con CamScanner176 — Satmonelta TABLE 9.2, Characteristics of Salmonella on Isolation and Biochemical Identification Media Salmonella Medium and = Other (Color) Reaction Interpretation Microorganisms Bismuth sulfite Colonies black to Salmonella survives the Most are inhibited; agar: light green with or strong selectivity of the E. coli may produce grey-green without dark halo medium; production of brown to green colonies and metallic sheen Colonies greenish blue with black green-yellowish center Xylose lysine Red with black desoxycholate centers agarired Triple sugar Slant: red (alkaline) iron agar: red Butt: yellow (acid) Black precipitate Lysine iron Slant: purple agar: purple (alkaline) Butt: purple (alkaline) Black precipitate HS No lactose, sucrose, or salicin fermentation; production of HS Limited carbohydrate fermentation; lysi decarboxylation; production of HS No significant acid from carbohydrates; alkaline products from protein breakdown Glucose fermentation Production of HS No acid from carbohydrates; alkaline products from protein breakdown Alkaline products from lysine decarboxylation Production of HS ‘Yellow to orange colonies (carbohydrate fermentation) Yellow (carbohydrate fermentation); red with no black center (no HS production) Slant: yellow (acid) Butt: red (alkaline) No precipitate Slant: yellow (acid) or red (neutral) Butt: yellow (acid) or red (neutral) No precipitate tion of the carbohydrates (lactose, salicin, and sucrose) in the medium. Acid pro- duction by sugar fermentation changes the color of the pH indicator to yellow, and thus colonies become surrounded with yellow agar. Salmonella does not ferment these sugars but uses the proteose peptone as a source of energy with the produc- tion of alkaline end products. Colonies of Salmonella, therefore, appear greenish blue as the result of the indicator’s color change. Ferric ammonium citrate and sodium thiosulfate combine to detect the production of desulfhydrase enzymes by Salmonella, In this case, the bacterial enzyme converts thiosulfate ($,0;*) to HLS, which can then react with the ferric ion (Fe™) to create a black precipitate. Salmonella colonies on HE agar appear greenish blue with a black center. Lactose Broth (LB) Lactose broth is a nonselective medium. The lactose in the medium is not usable by Salmonella, but a lactose-containing medium seems to promote the slow recovery Escaneado con CamScannerCONVENTIONAL METHOD 177 1 I ' | Slant reaction ; tic ‘Slant reaction - i ear Genatona pean’ | | by deamination of prtsin eamination of protein deamination of (typical for Salmoneta) | | (opi nel) on \ on a Yellow: Acidic (A) pH by | Yetlow Ace () eH by excessive sugar sxcantve oy fermentation 1 ' H,S production seen as 5 production HS production seen as | | HS on Fes (black ppt) | jut reaction Butt 1 Purple: Alkaline (K) pH ine Yellow: Acidic (A) pH by | | Dyn on (ypal sugar fermentation i acaronyaton (ye \ on | ‘Yellow: Acidic (A) pH by excessive sugar 1 fermentation Tsl agar ) uA Typical Salmonelia reactions: | Tele! Salmon reactions: KUA* (re slantiyliow butt with aK (purple slanvpurplo butt back ppt) H with black ppt) Fig. 9.3. ‘Typical Salmonella biochemical reactions on TSI agar and LIA. of injured Salmonella cells. Use of a broth medium dilutes the toxic or inhibitory Substances in the food, The non-Salmonella populations increase considerably Guring incubation of the LB-food mixture, The numbers of salmonellae will also fnerease, but at a slower rate than that experienced by most other species present. Lysine Iron Agar (LIA) ‘Thisisa differential agar medium that dispensed intubes to form aslant witha deep Thisis (hig 0.3). Presumptive Salmonella isolates are streaked on the slant and ott OM fn the tube but for biochemical identification (Fig. 9.3).Reactionsinthe slant stabbed i robie while those in the butt area are primarily anaerobic. The aerobic con- area a1® he slant is assured by Ieaving the tube cap only lightly tightened. dition of dium contains a limited amount of glucose, an ample supply of lysine, ands pit indicator (bromeresol purple). This pET indicator turns yellow in acidic Escaneado con CamScanner178 Salmoneia | cnvironment, red at neutral conditions, and purple under alkaline conditions. Salmonella quickly utilizes the glucose first and turns the medium yellow because of acid production, After depletion of glucose, Salmonella utilizes lysine as an energy source. Salmonellae possess the enzyme lysine decarboxylase and a decar- | boxylation reaction occurs under anaerobic conditions (tube butt). The decar- boxylation of lysine results in amine byproducts which produce alkaline reactions. The quantity of alkaline products is sufficient to neutralize the acid from glucose utilization and produce alkaline reaction in the tube butt (purple). In the aerobic slant, peptone metabolism is possible, When peptone is metabolized, this results in basic end products. These products turn the medium basic and thus a purple slant is observed. These fermentations may result is gas production in the LIA agar tube. If the gas bubbles are large enough, cracks can be formed, even breaking the agar into chunks. Salmonella may or may not generate enough gas to produce notice- able bubbles. A differential system that consists of sodium thiosulfate and ferric ammonium citrate is included in this medium. Hydrogen sulfide results from the activity of Salmonelta’s desulfhydrase on the thiosulfate, Released hydrogen sulfide reacts with the ferric ion, producing a'black precipitate, particularly around the stab. Selenite Cystine (SC) Broth ‘This medium is used for the selective enrichment of Salmonella. The selective agent, sodium acid selenite, is deleterious to most bacterial species. Salmonella, however, is more resistant to this chemical than are many other bacteria (such as fecal col- iforms and enterococci). Because of this resistance, the medium is used to selec- tively enrich food samples in Salmonella. x-Cystine is added to the medium as a reducing agent, Tetrathionate (TT) Broth Like SC broth, this medium also is used for the selective enrichment of Salmonella. Iodine in the medium catalyzes the conversion of sodium thiosulfate to tetrathion- ate (S,0,*—I, > $,0¢"). Tetrathionate is toxic to many bacteria. Salmonellae are selected for because they possess an enzyme, tetrathionate reductase, that detoxi fies the tetrathionate, Because salmonellae can detoxify the compound, they will multiply more rapidly in this medium than will other species. Bile salts in the medium also provide selection against nonenterics. Triple Sugar Iron (TSI) Agar ‘This is a differential medium prepared as a slant agar in test tubes (Fig. 9.3). The presumptive Salmonella isolates are streaked on the surface of the slant and stabbed in the butt of tube contents. Reactions in the slant area are aerobic, and those in the butt are primarily anaerobic. When this medium is used, the tube cap fitting should be loosened. If serew-cap tubes are used, the caps must be tightened loosely to maintain aerobic conditions in the slant portion. The medium contains three sugars, glucose, sucrose, and lactose, at 0.1, 1.0, and 1.0% levels, respectively. A pH indicator (phenol red) is included in the medium to Escaneado con CamScannerconventionaweT4on 179 detect acid production from fermentation of these carbohydrates. Phenol red changes medium color to yellow upon acid production by the inoculated microor- ganism while no color change indicates an alkaline surrounding. Salmonella metab- Olizes the limited supply of glucose (but not the sucrose or lactose) and produces acid in the relatively anaerobic butt of the TSI agar tube. Salmonella on the slant metabolizes glucose aerobically with no or little acid production. Additionally, aerobic breakdown of proteins by Salmonella results in basic end products that turn the slant alkaline. Microorganisms that metabolize lactose and sucrose produce strong acid reactions that turn the tube contents to yellow. Microorganisms may also produce carbon dioxide as a result of sugar metabolism. This results in bubbles in the agar. If the bubbles are large enough, cracks can be formed, even breaking the agar into chunks. Salmonella may or may not generate enough gas to produce noticeable bubbles. ‘The medium contains an additional differential system that consists of sodium thiosulfate (Na;S;O;) and ferrous sulfate. Salmonella’s desulfhydrase enzyme con- verts the thiosulfate into hydrogen sulfide, which reacts with ferrous sulfate (FeSO.), producing a black precipitate of ferrous sulfide (FS). This precipitate may “cloud” the entire butt region of the tube, making interpretation of butt reactions difficult, particularly when tubes are inadvertently incubated for longer than 24hr. Xylose Lysine Desoxycholate (XLD) Agar “This is a selective-differential medium used for the isolation of potential Salmonella colonies. The desoxycholate inhibits nonenterics. This medium uses ferric ammo- nium citrate and sodium thiosulfate as differential agents in the same way as does HE agar, producing the same possible result. A pH indicator (phenol red) is used to detect the fermentation of sugars (xylose, lactose, and sucrose) in the medium. Fermentation, and therefore acid production by these carbohydrates, turns this medium yellow. Of these sugars, Salmonella ferments only xylose. t-Lysine is addéd to the medium to differentiate salmonellae from other xylose fermenters. Salmonellae have the ability to decarboxylate lysine. Because Salmonella ferments 2 limited amount of sugar (only the xylose), the decarboxylation reaction results in sufficient basic products to balance and overwhelm the acidic products of xylose fermentation. This alkaline reversion turns the colonies and medium around them red. Therefore, the combination of pH indicator, sugars, and amino acid allows dif- ferentiation of organisms by metabolic activity. Salmonella colonies on XLD agar, therefore, are red with black centers. ORGANIZATION Each pair of students will test one food sample. Retail packages of ground meat (chicken, turkey, beef, or pork) will be tested for Salmonella using the Conventional/BAM protocol. TWo rapid methods, enzyme-linked immunosorbent gssay (ELISA) and DNA-based probe, may also be run concomitantly with the conventiona/BAM method. Escaneado con CamScanner180 Satmonena Period 1 Preenrichment MATERIALS AND EQUIPMENT Per Pair of Students = + Food sample i. + Culture of Salmonella (positive control) + Lactose broth a. One 225-ml flask for food enrichment b. One 10-ml tube for Salmonella enrichment (control) 3 Class Shared * Scale for weighing food samples (eg, a top-loading balance with 500g capacity) * Incubator, set at 35°C | + Stomacher and stomacher bags | + Container for holding enrichment bags z x 9, ZE BBB EBEBRPBUE@wuaZxuns PROCEDURE Control 1. Transfer 1ml of the Salmonella culture to the 10-ml lactose broth tube. 2. Incubate the inoculated lactose broth tube at 35°C for 24hr. The incubated mixture in these tubes will be referred to as “control-LB enrichment.” Food Sample 1. Prepare the food for sampling to ensure that a representative sample is analyzed. This varies with the food and may involve cutting, partitioning, grinding, or mixing. 2. Weigh 25g of the food sample into a stomacher bag, Add the 225ml lactose broth. Stomach for 2min, 3, Place the enrichment-containing bag in the tub or beaker designated by the laboratory instructor. This container should support the bag and keep it upright during incubation and handling. 4, Incubate the bags at 35°C for 24hr. The incubated mixture i be referred to as “food-LB enrichment.” these bags will | Escaneado con CamScanner“” SELECTIVE ENRICHMENT — 181 Period 2 Selective Enrichment MATERIALS AND EQUIPMENT Per Pair of Students + Control-LB enrichment + Food-LB enrichment + Two 10-ml tubes selenite cystine broth + Two 10-ml tubes tetrathionate broth Class Shared + Incubator, set at 35°C PROCEDURE Positive Control 1, Label one selenite cystine and one tetrathionate broth tube for the positive control. 2. Mix the content of the control-LB enrichment tube and transfer 1ml into each of the selenite cystine and tetrathionate broth tubes. 3. Incubate the inoculated tubes at 35°C for 24hr. The contents of the incubated ‘tubes will be referred to as “control-SC enrichment” and “control-TT enrich- ment,” respectively. Food Sample 4. Label one selenite cystine and one tetrathionate broth tube for the food sample. 2. Mix the food-LB enrichment by hand agitating the stomacher bag. 3, ‘Transfer 1ml food-LB enrichment into the selenite cystine broth tube; repeat with the tetrathionate broth tube. 4, Incubate the inoculated tubes at 35°C for 24hr. The contents of the incubated tubes will be referred to as “food-SC enrichment” and “food-TT enrichment,” respectively. Escaneado con CamScanner| 182 Saimonelta Period 3 Isolation on Selective Agar Media MATERIALS AND EQUIPMENT Per Pair of Students + Control-SC and control-TT enrichment tubes + Food-SC and food-TT enrichment tubes + Bismuth sulfite agar (two plates) + Hektoen enteric agar (two plates) + Xylose lysine desoxycholate agar (two plates) Class Shared + Incubator, set at 35°C PROCEDURE 1, Label one BS plate for the food sample and the other for the positive control. Divide each plate into halves, one for SC enrichment as the source and the other for the TT enrichment as the source. Repeat for the HE and XLD plates. 2. Two-phase streak the food-SC enrichment onto the appropriate half of the BS, HE, and XLD plates. Two-phase streak the food-TT enrichment onto the other half of the BS, HE, and XLD plates. 3. Repeat step 2 using the positive control (control-SC and control-TT enrichments). 7 4, Incubate the streaked plates at 35°C for 24hr. Escaneado con CamScanner+ BIOCHEMICAL IDENTIFICATION 183 Period 4 Biochemical Identification MATERIALS AND EQUIPMENT Per Pair of Students * BS, HE, and XLD incubated plates (prepared during the previous laboratory period) + Lysine iron agar slants (seven tubes) + Triple sugar iron agar slants (seven tubes) Class Shared + Incubator, set at 35°C PROCEDURE Inspection of BS, HE, and XLD Plates BS Agar Positive Control 1, Observe the BS plate inoculated with the positive control. Typical Salmonella colonies are black to green with or without a dark halo and metallic sheen. 2. Compare the appearance of colonies originating from the SC and TT enrich- ments, Note any differences in appearance or prevalence, 3. Record the observations in Table 9.3, Food Sample 1. Observe the BS plate inoculated with the enriched food sample. Compare the appearance of these colonies with that of the positive control. Mark the loca- tion of possible isolated colonies of Salmonella, 2. Compare the appearance of colonies originating from the SC and TT enrich- ments. Note any differences in appearance or prevalence. 3. Record the observations in Table 9.3, HE Agar Positive Control 1. Observe the HE plate inoculated with the positive control.'Iypical Salmonella colonies are greenish blue with black center, 2. Compare the appearance of colonies originating from the SC and ‘TT enrich- ments. Note any differences in appearance or prevalence, 3. Record the observations in Table 9.3. Food Sample 1, Observe the HE plate inoculated with the entiched food sample. Compare the appearance of these colonies with that of the positive control. Mark location of possible isolated colonies of Salmonella. a Escaneado con CamScanner(Write a descriptive title, including source of inoculum, previous manipulations, and results presented) Plating Enrichment ‘Appearance Medium Medium Food Sample Positive Control BS sc ar sc TT XLD sc 1T (Add at least five foomotes). 2. Compare the appearance of colonies originating from the SC and TT entich- ments. Note any differences in appearance or quantity, 3. Record the observations in Table 9:3, XLD Agar Positive Control 1. Observe the XLD plate inoculated with the positive control. ‘Typical Salmonella colonies are red-pink colonies. These colonies commonly have a black center. 2. Compare the appearance of colonies originating from the SC and TT enrich- ments, Note any differences in appearance or quantity. 3. Record the observations in Table 9.3. Food Sample 1. Observe the XLD plate inoculated with the enriched food sample. Compare the appearance of these colonies with that of the positive control. Mark the location of possible isolated colonies of Salmonella. 2. Compare the appearance of colonies originating from the SC and TT enrich- ments. Note any differences in appearance or quantity. 3. Record the observations in Table 9.3. Biochemical Identification 1, Label six tubes of each slant medium (TSI agar and LIA) for use with the enriched food sample. The label should include the medium from which the colony was obtained (BS, HE, XLD) and the type of enrichment (SC, TT), EE Eee Escaneado con CamScannerwo rs w BIOCHEMICAL IDENTIFICATION 185, Label one tube of TSI agar and one of LIA for the positive control. There will be a total of seven tubes of each medium. . Choose and mark two possible Salmonella colonies, if any are present, from each of the plates (BS, HE, and XLD) that were inoculated from the enriched food sample. One should be from the SC half and the other from the TT half. ‘This is a total of six colonics. . For each chosen colony, inoculate both a TSI agar and an LIA slant. a. Using a sterile inoculating needle, lightly touch the center of the selected colony. b. Inoculate the agar slant by beginning at the base of the slant and working upward in a zigzag pattern. Do not streak higher than two-thirds of the way up. c. Next, without flaming the loop, stab the needle into the butt, stopping 1-2cm from the base of the tube. . Inoculate one TSI agar and one LIA slant from any representative colony of the positive control. . Incubate the inoculated slants at 35°C for 24 br. Escaneado con CamScanner